Isoflurane anesthesia induced persistent, progressive memory impairment, caused a loss of neural stem cells, and reduced neurogenesis in young, but not adult, rodents

Abstract

Isoflurane and related anesthetics are widely used to anesthetize children, ranging from premature babies to adolescents. Concerns have been raised about the safety of these anesthetics in pediatric patients, particularly regarding possible negative effects on cognition. The purpose of this study was to investigate the effects of repeated isoflurane exposure of juvenile and mature animals on cognition and neurogenesis. Postnatal day 14 (P14) rats and mice, as well as adult (P60) rats, were anesthetized with isoflurane for 35 mins daily for four successive days. Object recognition, place learning and reversal learning as well as cell death and cytogenesis were evaluated. Object recognition and reversal learning were significantly impaired in isoflurane-treated young rats and mice, whereas adult animals were unaffected, and these deficits became more pronounced as the animals grew older. The memory deficit was paralleled by a decrease in the hippocampal stem cell pool and persistently reduced neurogenesis, subsequently causing a reduction in the number of dentate gyrus granule cell neurons in isoflurane-treated rats. There were no signs of increased cell death of progenitors or neurons in the hippocampus. These findings show a previously unknown mechanism of neurotoxicity, causing cognitive deficits in a clearly age-dependent manner.

Figure 1

Effects of isoflurane on physiologic parameters. (A) A representative real-time blood pressure curve. (B) The mean arterial blood pressure (mm Hg) was recorded from 15 mins after isoflurane induction and surgical insertion of the catheter, and then every 5 mins, during the first isoflurane exposure or during the fourth isoflurane exposure in young rats (n=6/group). The blood pressure was stable during isoflurane inhalation and there was no significant difference between the first and the fourth isoflurane exposure. (C) Rectal temperature was recorded from 15 mins after isoflurane induction. The temperature was stable during maintenance with isoflurane in P14 rat pups (n=8). (D) The blood parameters after isoflurane exposure showed a slight respiratory acidosis and hyperglycemia at both ages, compared to the controls (n=6/group). Four daily isoflurane exposures in the pups (P17) isoflurane did not induce any differences compared with single exposure. Data are shown as mean±s.e.m. ^*P<0.05, ^**P<0.01 compared with P14 control; ^¶P<0.05 compared with P60 control.

Figure 2

Isoflurane inhalation impairs object recognition in rats. (A) Experimental design for the postnatal day 14 (P14) and P60 rats. The rats were exposed to isoflurane or the mixture of oxygen and air for 35 mins daily for four successive days. Object recognition was tested either 4 weeks after the last isoflurane exposure or even later. (B) Recognition memory index was reduced significantly 4 weeks after isoflurane exposure in P14 to P45 rats (n=6 /group), but not P60 to P91 rats (n=7/group) (left panel). The recognition memory index reduction was even more pronounced 10 weeks after isoflurane exposure in P14 to P90 rats (n=7/group) (right panel), but not P60 to P120 (n=7/group).

Figure 3

Isoflurane inhalation impairs reversal learning in mice. (A) Experimental design for P14 mice exposed to isoflurane (n=16) or controls (n=15) for the IntelliCage test. (B) Incorrect visit ratio (%). (C) Number of correct visits. (D) Number of incorrect visits. (E) Average correct visit duration (secs). (F) Average incorrect visit duration (secs). (G) Number of correct nose pokes. (H) Number of incorrect nose pokes. (I) Number of nose pokes per correct visit. Values are presented as group mean±s.e.m per day (d) during learning and reversal learning. (^*Treatment, treatment × time. P<0.05, ^**/^##P<0.01, ^***/^###P<0.001.)

Figure 4

The total number of granule cell layer (GCL) neurons and the GCL volume. The total number of neurons in the GCL of the dentate gyrus was counted after DAPI staining using stereological principles 4 weeks after isoflurane inhalation (A) or after an even longer interval (B) for both P14 and P60 rats. The total number of GCL neurons decreased significantly 10 weeks after isoflurane inhalation in P14 rats (P14 to P90) compared with the controls (n=7/group) (^*P<0.05) (B), but not in the P14 to P45 group (n=6/group) (A). The P60 rats did not display lower neuronal numbers in any group (A, B). The GCL volume and the density of GCL neurons were not significantly different between isoflurane and control groups for any of the ages or intervals (C) (P60 to P91, P60 to P120, n=7/group).

Figure 5

Cell death-related markers in the granule cell layer (GCL) and the CA1 24 h after the last isoflurane inhalation. (A) Representative AIF, active caspase-3, FBDP, LC3, and TUNEL immunopositive cells in the GCL 24 h after the last isoflurane inhalation in P14 rats. (B) The density of immunopositive cells in the GCL (including the subgranular zone) and CA1 did not show any significant differences between the control and isoflurane groups. (C) A representative TUNEL (green) and BrdU (red) staining in the GCL. (D) The density of TUNEL and BrdU double-positive cells in the GCL was not different between the two groups (n=6/group).

Figure 6

Synapsin I immunoreactivity in the hippocampus. (A) P14 rats were exposed to isoflurane for 35 mins daily for four successive days and killed at P90 (n=7/group). (B) A representative synapsin I immunostaining showing different cellular layers in the DG and CA3. (C) The optical density of synapsin I immunoreacitivity in the DG and CA3 was quantified in three sections per animal and the average number was used as n=1 for each animal. There were no significant differences in any of the measured layers between the two groups. GCL, granule cell layer; IML, inner molecular layer; MML, middle molecular layer; OML, outer molecular layer; SR, stratum radiatum; SL, stratum lucidum; SO, stratum oriens.

Figure 7

Isoflurane reduced cell proliferation and neurogenesis in the immature rat GCL. (A) Experimental design for both P14 and P60 rats. BrdU was injected immediately after each isoflurane inhalation (35 mins/day) and rats were killed either 1 day or 4 weeks after the last exposure. (B) Representative BrdU and phospho-histone H3 (P-H3) stainings in the GCL (left panel) and quantification of BrdU-positive (middle panel) and P-H3-positive cells (right panel). BrdU- and P-H3-positive cells were quantified by using stereological principles. BrdU-labeled cells were reduced by 21% at 24 h and P-H3-labeled cells were reduced by 71% at 4 weeks after the last isoflurane inhalation in P14 rats (P14 to P18, P14 to P45, n=6 /group). Adult rats displayed no differences (n=8 /group for P60 to P64 group, n=7/group for P60 to P91 group). (C) Representative BrdU stainings from P14 to P45 and P60 to P91 rats and triple labeling of BrdU (green), NeuN (red), and S-100β (blue) in the GCL. (D) The bar graphs indicate that isoflurane decreased BrdU incorporation in the P14 to P45 group (left panel) and neurogenesis in both the P14 to P45 (n=6/group) and the P60 to P91 (n=7/group) groups, but more pronounced in P14 to P45 rats (middle panel). Astrocyte formation increased significantly in both groups (right panel). ^*P<0.05, ^**P<0.01.

Figure 8

Isoflurane reduced the neural stem cell pool in the immature rat GCL. (A) A confocal image showing a representative SOX-2 (green) and GFAP (red) double labeling in the GCL. Scale bar=20 μm. (B) The numbers of undifferentiated neural stem cells (SOX-2/GFAP double-positive cells) were counted in the entire GCL and the bar graph shows a significant decrease in the number of stem cells in the immature rat GCL after isoflurane exposure (n=6/group), but not in the adult rat GCL (n=7/group). ^***P<0.001.